The human CD8+ T cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes

Abstract

The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8(+) T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8(+) T cell responses after live attenuated dengue vaccination and show that CD8(+) T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.

Importance: The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that seroconversion is the only reliable correlate of protection. Here, we show that CD8(+) T cell responses in vaccinees were readily detectable and comparable to natural dengue virus infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.

FIG 1

Protein location of epitopes varies as a function of the DENV serotype. (A) Study participants were vaccinated with one of the four live attenuated dengue vaccine candidates. PBMC from each donor were tested against peptides predicted for the exact strain with which they were vaccinated. Responses detected in study participants vaccinated with DEN1Δ30 (B), DEN2/4Δ30 (C), DEN3Δ30/31 (D), or DEN4Δ30 (E) live attenuated monovalent dengue vaccine candidates are shown. Our stringent criteria of positivity require consistent responses in two out of two independent experiments. Responses are expressed as the number of IFN-γ-secreting cells per 10⁶ PBMC and considered positive if the magnitude of response to the test peptide was significantly different compared to a negative-control peptide (P < 0.05, Student t test) and the stimulation index (SI; SI = ratio test SFC/control SFC) was >2.0.

FIG 2

The immune response induced by tetravalent vaccination is targeted against highly conserved proteins and displays a multifunctional effector memory phenotype. (A) Responses detected in study participants vaccinated with tetravalent DLAV (TV003) are shown. Responses are expressed as the number of IFN-γ-secreting cells per 10⁶ PBMC. (B) Analysis of conservancy of the epitopes identified. The relative response by epitopes derived from serotype-specific regions (white pie chart) or regions conserved between serotypes (black pie chart) is shown. The right panel shows the number of serotypes in which the responses are conserved. (C) Gating strategy for phenotype and cytokine profiling of responding cells. (D and E) PBMC from tetravalent vaccinees (n = 4) were incubated with HLA-matched epitope pools and assayed for the production of IFN-γ and TNF-α as described in Materials and Methods. Each sample was stained additionally with antibodies against CD45RA and CCR7 to determine the proportion of cells in the following subsets: CCR7⁻ CD45RA⁻ T_EM (effector memory T cells), CCR7⁺ CD45RA⁻ T_CM (central memory T cells), CCR7⁺ CD45RA⁺ T_N (naive T cells), and CCR7⁻ CD45RA⁺ T_EMRA (effector memory T cells reexpressing CD45RA). The relative distribution of T cell subsets within the IFN-γ positive cells is shown (D), as well as CD8⁺ T cells positive for one of the cytokines (E; IFN-γ, ●; TNF-α, ◼) or double positive for both cytokines (E; ▲). Stimulated frequencies are shown as net frequencies with the baseline levels subtracted (cytokine production of stimulated CD8⁺ T cells minus that of unstimulated CD8⁺ T cells).

FIG 3

Epitopes induced by DLAV are highly conserved in field isolates of DENV and recognized by donors exposed to natural infection with DENV. (A) Conservancy of epitopes identified within field isolates of DENV. Totals of 162 DENV1, 171 DENV2, 169 DENV3, and 53 DENV4 sequences were retrieved from the NCBI protein database. The relative conservancy within the sequences of the respective serotypes for all epitopes identified after monovalent (left panel) or tetravalent vaccination (middle panel) is shown. The right panel shows all epitope reactivity to either the monovalent or tetravalent vaccination. (B and C) Phenotype (B) and cytokine (C) profiles of T cell responses in blood donors previously exposed to natural secondary infection with DENV epitopes. PBMC samples (n = 4) were incubated with HLA matched vaccine-specific epitope pools and assayed for production of IFN-γ and TNF-α as described in Materials and Methods. Each sample was also stained with antibodies against CD45RA and CCR7 to determine the proportion of cells in the following subsets: CCR7⁻ CD45RA⁻ T_EM (effector memory T cells), CCR7⁺ CD45RA⁻ T_CM (central memory T cells), CCR7⁺ CD45RA⁺ T_N (naive T cells), and CCR7⁻ CD45RA⁺ T_EMRA (effector memory T cells reexpressing CD45RA). Responses were detected in three out of four donors tested. Shown is the relative distribution of T cell subsets within the IFN-γ-positive cells (B) and the relative distribution of cells positive for one of the cytokines (C; IFN-γ, ●; TNF-α, ◼) or double positive for both cytokines (C; ▲).